The purpose of this proposal is to better understand the mechanism underlying the process of multicellular assembly exhibited by cranial neural crest cells during facial morphogenesis. It is proposed that this process occurs by a self-assembly mechanism and, further, that the biochemical differences observed between cells after mosaic formation, arose as a consequence of the assembly process. To test this hypothesis the eukaryotic microorganism Dictyostelium discoideum was chosen for studies. This organism forms a multicellular aggregate from genetically, biochemically and cytologically identical cells at a specific stage in its development. Since these cells migrate and aggregate in response to a chemotactic signalling system involving cAMP, the possibility exists that this system is the mechanism regulating cellular differentiation. To better understand this signalling system, experiments on the D. discoideum adenylate cyclase are described. Experiments based on results obtained during the previous project period are presented. These experiments, designed to further characterize the loss of cyclase catalytic activity, include comparative studies of the response of both cytoplasmic and solubilized cyclase, the nucleotide specificity, requirement for membrane structural components, and the effect of polyamines. The developmental significance of this effect will be studied with membranes prepared from cells at different growth stages and also from mutants. In addition, experiments are planned to explore the mechanism underlying the ATP induced loss of catalytic activity. One enzymatic reaction to be examined in some detail is the adenyl transfer activity. The product of this reaction is to be isolated, and characterized. Further, the role of the 5'-nucleotide phosphodiesterase in the deadenylylation activity will be studied using 4-nitrophenyl phenylphosphate. The effect of this phosphonate ester on adenylylation product formation, AMP formation and finally on adenylate cyclase activity will also be studied. Studies on the 4-nitrophenyl phenylphosphate activity itself will also be undertaken. These include isolation and purification of the enzyme, its developmental regulation, and finally a comparison of this activity wth the specific 3',5'-cyclic phosphodiesterase. Finally, modulation of the ATP effect will be studied using the pulsed cAMP addition technique.